Microencapsulation of living tissue and cells

ABSTRACT

Living tissue or cells, for example, islets of Langerhans, are microencapsulated for implantation in the body for long term treatment of diabetes or other disease requiring organ transplantation. The microcapsules take the form of a biocompatible semi-permeable hydrogel membrane which permits the passage of materials and oxygen to the cells and metabolic products from the cells while retaining the cells encapsulated. The biocompatible semi-permeable membrane has an outer negatively-charged surface, which imparts to the microcapsules the ability to maintain long term effectiveness.

This is a continuation of application 4,689,293, which Ser. No. 677,985filed December 4, 1985, now U.S. patent of application Ser. No. 501,445filed June 6, 1983, now abandoned.

FIELD OF INVENTION

The present invention is concerned with the microencapsulation of livingtissue or individual cells.

BACKGROUND TO THE INVENTION

Various attempts have been made to microencapsulate biologically activemacromolecules, tissue and individual cells so that they remain viableand in a protected state within a semi-permeable membrane which permitspassage of low molecular weight substances, such as nutrients andoxygen, but not of high molecular weight substances, such as, proteinsand cells. However, none of these attempts has been successful inproviding microcapsules in which tissue or cells enclosed within thesemi-permeable membrane are able to survive in an animal body for longerthan 2 to 3 weeks, which severely limits the utility of the products inthe treatment of diseases requiring organ transplantation, such asdiabetes.

In "Semipermeable Microcapsules" by T.M.S. Chang, Science, 146, 1964,524 to 525, there is described the microencapsulation of erythrocytehemolysate and urease in semi-permeable polyamide (nylon) membranes.These microcapsules did not survive for long when injected into theblood stream. Papers have described the preparation of semi-permeablemicrocapsules containing microbial cells and viable red blood cells,namely K. Mosbach and R. Mosbach, Acta Chem. Scand., 20, 1966, 2807 to2812 and T.M.S. Chang, F. C. MacIntosh and S. G. Mason, "Semi-permeableAqueous Microcapsules", Can. J. Physiol. and Pharmacology, 44, 1966, 115to 128. The Chang et al article mentions for the first time thepossibility of using injections of encapsulated cells for organreplacement therapy.

The next significant development was the use of calcium and aluminumalginate gels for the immobilization of microbial cells and enzymes. Thecells were immobilized under extremely mild conditions, thus maintainingtheir viability. This work was described in V. Hackel, J. Klein, R.Megret and F. Wagner, Europ. J. Appl. Microbiol., 1, 1975, 291 to 296and M. Kierstan and C. Bucke, "The Immobilization of Microbial Cells,Subcellular Organelles, and Enzymes in Calcium Alginate Gels",Biotechnology and Bioengineering, 19, 1977, 387 to 397.

Subsequently, viable tissue and cells were immobilized in alginatedroplets coated with polylysine (F. Lim and R. D. Moss,"Microencapsulation of Living Cells and Tissues", J. Pharm. Sci. 70,1981, 351 to 354). While the cells remained viable in culture for up totwo months, no experiments are described to test the in-vivobiocompatibility of the polylysine membrane. At approximately the sametime, there was reported for the first time, the use ofmicroencapsulated islets to correct the diabetic state of diabeticanimals, in F. Lim and A. M. Sun, "Microencapsulated Islets asBioartificial Pancreas", Science, 210, 1980, 908 to 909. However, themicrocapsules, consisting of an inner alginate core, followed by apolylysine coat and an outer polyethyleneimine membrane, were rejectedby an animal body within 2 to 3 weeks of implantation due to the poorbiocompatibility of the outer polyethyleneimine membrane.

Formation of the latter microcapsules also is described in U.S. PatentNo. 4,352,883 F. Lim. As set forth therein, finely divided living tissueis suspended in an aqueous medium which contains sodium alginate, thesuspension is formed into droplets of a size to envelop tissue, thedroplets are gelled by conversion to calcium alginate to form discrete,shape-retaining temporary capsules, a permanent semi-permeable membraneis formed about the temporary capsules, and the calcium alginate gel isreliquified within the membrane by ion exchange Example 3 of the patentdescribes injection of the microcapsules into diabetic rats.Polyethyleneimine contains imino groups, which induce granuloma,resulting in an inflammatory response from the body, which, in turn,destroys the polymer. Polyethyleneimine, therefore, is not biocompatibleand the microcapsules are ineffective for organ replacement therapy fora period lasting longer than 2 to 3 weeks.

U.S. Pat. No. 4,352,883 mentions the possibility of using polylysine, amuch more biocompatible material, instead of polyethyleneimine as themembrane. Polylysine is positively charged and it is well known thatpositively-charged surfaces are excellent substrates for cell growth.Cell growth on the surface of the microcapsules, such as would occurwith a polylysine membrane, would transform the semipermeable capsularwall to an impermeable one, resulting in the death of the encapsulatedtissue.

It is apparent, therefore, that there is a need for the development ofmicrocapsules which can be implanted into an animal body and beeffective in the treatment of diseases requiring organ transplantation,such as, diabetes, for extended periods of time.

SUMMARY OF INVENTION

In accordance with the present invention, it has now surprisingly beenfound that living cells can be microencapsulated and the resultingmicrocapsules have long term in vivo activity by encapsulating the cellswithin a biocompatible semi-permeable membrane which has an outersurface of biocompatible negatively-charged material. The presentinvention, therefore, provides biocompatible microcapsules suitable forimplantation in a mammalian body comprising encapsulated viable tissueor individual cells within a biocompatible semi-permeable membranehaving a biocompatible negatively-charged surface. While the presentinvention has particular application to the microencapsulation of livingcells, any desired macromolecular core material may be provided in theform of microcapsules, such as, enzymes, immunoproteins and activatedcarbon particles. The macromolecular core material is surrounded by abiocompatible semi-permeable membrane which is permeable to smallmolecules for contact with the core material but is impermeable to thecore material, and also to potentially deleterious large molecules.

GENERAL DESCRIPTION OF INVENTION

In the present invention, core material, such as, living tissue,individual cells or biologically-active materials, are encapsulated in abiocompatible semi-permeable membrane, in the form of a hydrogel. Thematerial to be encapsulated is suspended in a physiologically-compatiblemedium containing a water soluble substance which can be reversiblygelled to provide a temporary protective environment for the tissue. Themedium is formed into droplets containing the tissue and gelled, forexample, by changing conditions of temperature, pH or ionic environment,to form temporary capsules, preferably of substantially perfectspherical shape so as to provide an overall improved physical strengthwhen compared with microcapsules formed from non-spherical capsules.Thereafter, the temporary capsules which result are treated to form amembrane of controlled permeability about the shape-retaining temporarycapsules. The semi-permeable nature of the membrane permits nutrientsand oxygen to flow to the core material and metabolic products to flowtherefrom while retaining the core material within the microcapsule. Thebiocompatible nature of the semi-permeable membrane allows the passageof such materials to and from the core to occur without inflammation orother adverse body response while the outer negatively-charged surfaceinhibits surficial cell growth, so that the membrane remainssemi-permeable and effective for extended periods of time, typicallyfrom three to six months or longer.

The temporary capsules may be formed from any non-toxic water-solublesubstance that can be gelled to form a shape retaining mass by a changeof conditions in the medium in which it is placed, and also comprisesplural groups that are readily ionized to form anionic or cationicgroups. The presence of such groups enables surface layers of thecapsule to cross-link to produce a permanent membrane when exposed topolymers containing multiple functionalities of the opposite charge.

Preferably, the temporary capsules are formed from a polysaccharide gum,which may be natural or synthetic, of a type that can be gelled to forma shape retaining mass by exposure to a change in conditions and can bepermanently cross-linked or hardened by polymers containing reactivegroups, such as amino groups, which can react with the acidicpolysaccharide constituents. Most preferably, the gum is alkali metalalginate, specifically sodium alginate, although other water-solublegums may be used.

The temporary capsules may be formed from sodium alginate by extrudingdroplets of aqueous sodium alginate solution into an aqueous calciumchloride solution. As noted above, it is preferred that the temporarycapsules be substantially spherical and it has been found thatsubstantially perfectly spherical temporary capsules can be formed byusing an aqueous sodium alginate solution having a viscosity of at leastabout 30 centipoise. At viscosities below this critical lower limit, thetemporary capsules have an irregular shape.

Perfectly spherical capsules are obtained over a wide range of viscosityof the sodium alginate solution, with an upper limit being dictatedlargely by the ability to extrude the solution into the hardeningmedium. Usually, the viscosity of the aqueous sodium alginate solutiondoes not exceed about 1000 cps.

Formation of the permanent semi-permeable membrane about the temporarycapsules preferably is effected by ionic reaction between free acidgroups in the surface layer of the gelled gum and biocompatible polymerscontaining acid-reactive groups, such as, amino groups, typically in adilute aqueous solution of the selected polymer.

Cross-linking biocompatible polymers which may be used includepolylysine and other polyamino acids. It is noted that polyethyleneimineand other imine-containing polymers are unsuitable for membraneformation in view of the non-biocompatible nature. The molecular weightof the polyamino polymer may vary widely, depending on the degree ofpermeability required, and typically is in the range of about 11,000 toabout 400,000, preferably about 11,000 to about 100,000. The use ofpolylysine or other polyamino acid results in microcapsules having apositively-charged surface, which, as already noted, would be unsuitablefor long term viability, although the microcapsules are biocompatible.

In accordance with the present invention, the semi-permeable membrane istreated with a non-toxic biocompatible water-soluble polymeric materialwhich is capable of ionic reaction with free amino groups to form anouter negatively-charged coating about the membrane, typically bysuspension of the microcapsules in an aqueous solution of the polymericmaterial. The material used to form the outer coating preferably is thesame material as is used to form the temporary capsules, preferably apolysaccharide gum, more preferably an alkali metal alginate, such as,sodium alginate. Other biocompatible polymeric materials containingbase-reactive groups, such as, polyvinyl alcohol, polylactic acid, polyglycolic-lactic acid copolymers and poly beta-hydroxy butyric acid, maybe used to form the outer coating to the microcapsules. Molecularweights of such polymeric materials typically vary from about 10⁴ toabout 10⁶.

The treatment of the polyamino microcapsules with the biocompatiblebase-reactive material retains the overall biocompatible nature of thesemi-permeable membrane and, more importantly, results in anegatively-charged outer surface which inhibits cell growth and,therefore, permits the semi-permeable membrane to retain itspermeability and hence effectiveness over an extended period of time.

Following formation of the microcapsules, reliquification of thesuspending medium for the core material may be effected byre-establishing the conditions under which the material is liquid. Thismay be achieved by ion exchange to remove multivalent cation, forexample, by immersion in phosphate buffered saline or citrate buffer.

The process of the invention may be used to encapsulate living tissue,multicellular fractions thereof or individual cells, for example, isletsof Langerhans, liver cells and red blood cells, and otherbiologically-active material. The microcapsules which result may beimplanted into an appropriate site within a mammalian body for thepurpose of providing the body with the specialized physiologicalfunction of the tissue while the tissue remains viable. The implantationmay be achieved by simple injection, so that surgical procedures are notrequired.

The biocompatible semi-permeable membrane encapsulating the corematerial consists of interpenetrating layers of ionically-interactedbiocompatible materials. The overall wall thickness of thesemi-permeable membrane usually varies from about 5 to about 20 μm. Themicrocapsules themselves usually have a diameter in the range of about50 to about 2000 μm, preferably in the range of about 200 to about 1000μm for microcapsules containing islets of Langerhans as the corematerial. The biocompatible semi-permeable membrane is in the form of ahydrogel and hence has an overall water content within the membranestructure of at least about 20 wt %, which may vary up to about 90 wt %in the surface region.

The materials which are used to form the biocompatible semi-permeablemembrane are biodegradable by the body into which the microcapsules areimplanted. Such biodegradation takes place over the active life of themicrocapsules and is responsible for the ultimate failure of themicrocapsules. The biodegradation is a very slow process, as isevidenced by observed effectiveness of the control of blood sugar inrats by microencapsulated islets of Langerhans of at least three monthsand, in some cases, as long as one year.

DESCRIPTION OF PREFERRED EMBODIMENT

In a particularly preferred embodiment of the invention, living cellsare microencapsulated within a polylysine-alginate semi-permeablehydrogel by suspending cells uniformly in a sodium alginate solution inphysiological saline. Where the microcapsules are to be used for thetreatment of diabetes by controlling blood sugar in animals, includinghumans, the living cells take the form of islets of Langerhans from ananimal pancreas.

Spherical droplets containing the cells are produced from an aqueoussodium alginate solution by a droplet generator, such as, syringe pumpextrusion or electrostatic extrusion, and are collected as gelledspheres in a hardening solution, such as, calcium chloride. Themicrocapsules then are coated with polylysine followed by an outercoating of sodium alginate. The microcapsules may then be suspended inisotonic sodium citrate or other convenient ion exchange medium toreliquify the alginate gel inside the microcapsule.

The outer biochemically inert but biocompatible alginate surface is anegatively-charged hydrogel containing up to about 90% water. The lowinterfacial tension between the swollen gel surface and the aqueousbiological environment minimizes protein interaction, otherwise a strongprotein-polymer interaction may cause a severe inflammatory response.The biocompatibility of the hydrogel membrane leads to long termviability of the capsules when implanted. Polyethyleneimine-surfacedmicrocapsules do not appear to possess this property and hence arerejected by the body and produce a strong inflammatory response, whichseverely limits the useful life of the microcapsules within the body.The soft rubbery consistency of most hydrogels may also contribute totheir biocompatibility by decreasing frictional irritation tosurrounding tissues.

The durability of the microcapsules can be increased further byincreasing the thickness of the polylysine membrane, as compared withthe thickness of the polylysine-polyethyleneimine membrane used in U.S.Pat. No. 4,352,883. The strength of the microcapsules also may beincreased by cross-linking, for example, using glutaraldehyde, prior toreliquification of the gel.

In the present invention, it is not essential that the biocompatibleouter surface be composed of sodium alginate, but it is essential thatthe outer surface be biocompatible and negatively-charged. Bindingoccurs between the negatively-charged groups, usually hydroxyl orcarboxyl groups, and the positively-charged amino groups on polylysine.

The permeability of the microcapsule to nutrients and metabolic productsmay be varied by varying the molecular weight of the polylysine used informing the semi-permeable membrane. Usually, the molecular weight ofthe polylysine varies from about 11,000 up to about 400,000, preferablyabout 11,000 to about 100,000. Higher molecular weights lead to greaterpermeability than lower molecular weights.

EXAMPLES EXAMPLE 1

This Example illustrates the microencapsulation of islets of Langerhans.

Cultured rat islets of Langerhans (2×10³ islets in 0.2 ml medium) weresuspended uniformly in 2 ml of a 1.5% (w/w) sodium alginate solution(viscosity 51 cps) in physiological saline. Spherical dropletscontaining islets were produced by syringe pump extrusion through a22-gauge needle and collected in 1.5% (w/w) calcium chloride solution.The supernatant was decanted and the gelled spherical alginate droplets,containing islets, were washed with dilute CHES(2-cyclohexylamino-ethane sulfonic acid) solution and 1.1% calciumchloride solution.

After aspirating off the supernatant, the gelled droplets were incubatedfor exactly 6 minutes in 0.05% (w/w) polylysine having a molecularweight of 25,000. (These conditions are a significant increase inincubation time and polylysine concentration compared to the proceduresin U.S. Pat. No. 4,352,883, wherein Lim used 0.013% polylysine and 3minutes incubation time, and in the reported work of Lim and Sun wherethey used 0.02% polylysine and 2-5 minutes incubation time. Thesechanges result in a stronger polylysine membrane.)

The supernatant was decanted and the polylysine capsules were washedwith dilute CHES, 1 1% calcium chloride solution and physiologicalsaline. The washed polylysine capsules were incubated for 4 minutes in30 ml of 0.03% sodium alginate to permit the formation of an outeralginate membrane on the initial polylysine membrane, by ionicinteraction between the negatively charged alginate and the positivelycharged polylysine.

The resulting microcapsules were washed with saline, 0.05M citratebuffer for 6 minutes to reliquify the inner calcium alginate, and afinal saline wash The microcapsules were found to be perfectly sphericaland each to contain from 1 to 2 viable islets. The microcapsules haddiameters varying from 200 to 1000 μm and wall thicknesses varying from5 to 10 μm. The microcapsules were suspended in nutrient medium at 37°C.

The experiment was repeated with islet cells from mouse, bovine and dogpancreas and similar microencapsulated products were formed

EXAMPLE 2

This Example illustrates the viability of the microencapsulated islets.

In perifusion experiments, the insulin secretion from themicroencapsulated rat islets produced in accordance with the procedureof Example 1 was determined to be comparable with that fromunencapsulated islets. When the glucose concentration was raised from 50to 300 mg, there was a biphasic response of insulin release from bothgroups of islets and the insulin secretion increased.

The increase in the quantity of insulin in the presence of a highglucose concentration clearly demonstrated that the viability andfunctionality of the cells were retained throughout the process ofmicroencapsulation.

After 2 months in culture at 37° C., the microencapsulated islets wereobserved to have remained morphologically and functionally intact.

EXAMPLE 3

This Example illustrates the injection of microencapsulated islets intodiabetic rats.

Diabetic rats with blood glucose levels in the range of 370 to 470 mg/dLwere treated with approximately 3×10³ rat islets microencapsulated asset forth in Example 1. The microcapsules were introduced by injectioninto the peritoneal cavity using a 16-gauge needle fitted into asyringe.

Unencapsulated islets and islets microencapsulated in apolylysine-polyethyleneimine membrane, produced as described in U.S.Pat. No. 4,352,883 (Lim), were used as controls. Blood glucose levelswere assayed twice per week to determine the period of time for whichthe blood glucose level was lowered The results obtained are set forthin the following Table I:

                  TABLE I                                                         ______________________________________                                        Membrane           Number of Weeks Blood                                      Type               Glucose Level Lowered                                      ______________________________________                                        None               1        (N = 4)                                           Polylysine polyethyleneimine                                                                     2 to 3   (N = 8)                                           (Lim Patent)                                                                  Polylysine alginate                                                                              13 to 52  (N = 10)                                         (Present invention)                                                           ______________________________________                                    

As can be seen from the results of Table I, the islets enclosed in thebiocompatible polylysine alginate membranes of the invention survived upto 52 weeks, as demonstrated by the normal blood sugar levels in thediabetic rats. In contrast, the islets enclosed in thepolylysine-polyethyleneimine capsular membranes of the Lim Patent showedsurvival times of less than 3 weeks.

EXAMPLE 4

This Example shows the effect of multiple injections ofmicroencapsulated islets.

The procedure of Example 3 was repeated except that, following a returnto hyperglycemia (blood sugar concentration greater than 300 mg/dL), asecond injection of polylysine alginate microencapsulated isletsproduced in accordance with the procedure of Example 1 normalized theblood sugar level of the animal for a longer period than the initialinjections, allowing the blood sugar level of the diabetic rats to becontrolled for longer than six months with just two injections.

In contrast, five injections of polylysine-polyethyleneiminemicroencapsulated islets at 2 to 3 week intervals were barely able tocontrol the blood glucose level of diabetic animals for three months(N=8).

EXAMPLE 5

This Example illustrates the injection of microencapsulated rat isletsinto diabetic mice.

The procedure of Example 3 was repeated except that fewer islets wereused (1000 rat islets) and diabetic mice were employed. No polylysinepolyethyleneimine microcapsules were used as controls.

Blood sugar levels in the diabetic mice were controlled for more thantwo months with a single injection (I.P.), indicating that xenografttransplants (cross-species) are possible.

EXAMPLE 6

This Example illustrates the viability of recovered microencapsulatedtransplanted islets.

Microencapsulated islets were recovered from some of the treateddiabetic rats in Example 3 at 3, 5 and 12 months postimplantation. Themajority of the microcapsules were still physically intact and containedviable insulin-secreting islets, as demonstrated by secretion of insulinfrom the recovered islets in culture in response to a high glucoseconcentration.

EXAMPLE 7

This Example illustrates the microencapsulation of liver cells.

The procedure of Example 1 was repeated, except that liver cells wereemployed in place of islets An electrostatic droplet generator wasemployed in place of the syringe pump extruder to produce smallercapsules of diameter from 100 to 300 μm. Capsules containing viableliver cells were obtained, as determined by trypan blue exclusion and ahistological study. Each capsule was observed to contain about 300 livercells.

EXAMPLE 8

This Example illustrates the use of polyvinyl alcohol as the externalsurface of the microcapsules.

The procedure of Example 1 was repeated, except that 1.0% (w/w) solutionof polyvinyl alcohol in phosphate buffered saline was used in place ofhe sodium alginate solution for formation of the outer membrane coating.The polyvinyl alcohol did not significantly alter the permeability ofthe capsular membrane.

Polyvinyl alcohol is known to be a biocompatible water-soluble polymerand has been used in many surgical applications, such as,thromboresistant coatings for artificial blood vessels, and hence themicrocapsules produced in this Example are expected to exhibit similarblood sugar decreasing capability in diabetic animals to themicrocapsules produced by the procedure of Example 1.

EXAMPLE 9

This Example illustrates the use of polylactic acid as the externalsurface of the microcapsules.

The procedure of Example 1 was repeated, except that 0.1% (w/w) solutionof polylactic acid in buffered saline was used in place of the sodiumalginate solution for formation of the outer membrane coating. Thepolylactic acid was initially dissolved in dilute sodium hydroxide andthen neutralized with hydrochloric acid. The ongoing viability of theislets in the microcapsules so produced was demonstrated with trypanblue staining. Polylactic acid is a biocompatible polymer that iscurrently in clinical use as suture material. It is expected, therefore,that the microcapsules produced in this Example will exhibit similarblood sugar decreasing capability in diabetic animals to themicrocapsules produced by the procedure of Example 1.

EXAMPLE 10

This Example illustrates the preparation of spherical calcium alginatedroplets.

Sodium alginate solutions of varying concentrations (and henceviscosities) were extruded with a syringe pump through a 22 gauge needleinto a 1.5% (w/w) calcium chloride hardening solution and the resultinggel droplets were collected and their physical shape observed Theresults are reproduced in the following Table II:

                  TABLE II                                                        ______________________________________                                                               Fractions of Droplets                                  Sodium Alginate                                                                             Viscosity                                                                              which are Spherical                                    % (w/w)       (cps)    (%)                                                    ______________________________________                                        1.5           51       100                                                    1.4           43       100                                                    1.3           36       100                                                    1.2           30       100                                                    1.1           25       <25                                                    1.0           20        0                                                     0.9           16        0                                                     0.7           11        0                                                     0.3            4        0                                                     ______________________________________                                    

While in all instances, the droplets could be broadly described as"spheroidal", it will be apparent from Table I that it is only atconcentrations of sodium alginate solution of 1.2% w/w and above, i.e.viscosities of 30 cps and above, that perfect spheres are formed.

EXAMPLE 11

This Example illustrates variation of the microcapsule permeability.

The procedures of Examples 1, 8 and 9 were repeated, except that themolecular weight of the polylysine was varied, with microcapsules beingproduced from polylysine of molecular weight from 11,000 up to 400,000.The permeability of the resulting microcapsules was determined by thediffusion of serum albumin or ¹²⁵ I.Ig G (antibody) into and out of themicrocapsules.

It was found that the use of the 400,000 molecular weight polylysineincreased the permeability of the microcapsules while the use of the11,000 molecular weight polylysine decreased the permeability of themicrocapsules.

Capsules prepared using 0.075 wt % of mixed molecular weight polylysinein the process of Example 1, comprising 10 mg polylysine of 25,000molecular weight and 5 mg of polylysine of 4,000 molecular weight werefound to be less permeable to lysed red blood cells, when compared tocapsules prepared with 0.075 wt % of polylysine of 25,000 molecularweight.

It was further found that the microcapsules having a polylactic acidouter coating had a greater permeability than the alginate and polyvinylalcohol coated microcapsules at the same polylysine molecular weight.

The procedure of Example 1 was again repeated, except that theconcentration of polylysine was doubled to 0.1% w/w and the contact timewas doubled to 12 minutes, thereby increasing the thickness of thepolylysine layer from about 5 μm to about 20 μm. The resultingmicrocapsules exhibit decreased permeability when compared to thoseproduced in Example 1.

EXAMPLE 12

This Example illustrates increasing the strength of the microcapsules.

The procedures of Examples 1, 8 and 9 were repeated, except that themicrocapsules were placed in contact with 0.01% w/w glutaraldehyde forless than 60 seconds, just after the polylysine coating step or justbefore the citrate washing step. The microcapsules which result are moredifficult to break physically (using fine tweezers) and also are moredifficult to dissociate in a heparin solution, when compared withuncross-linked material.

SUMMARY OF DISCLOSURE

In summary of this disclosure, the present invention provides novelmicrocapsules of living tissue or cells which have long termbiocompatability and viability, and hence utility, in the treatment ofdiseases requiring organ transplantation, such as, diabetes.Modifications are possible within the scope of the invention.

What we claim is:
 1. A biocompatible in microcapsule, suitable forimplantation into an animal body and having a diameter of about 50 toabout 2000 μm, comprising:a macromolecular spherical core containingliving tissue or individual cells thereof, said core being surrounded byof interpenetrating layers of ionically-interacted biocompatiblematerials defining a membrane thickness of about 5 to about 20 μm, saidbiocompatible materials comprising a biocompatible polyamino acidpolymer normally having a positively-charged surface and a non-toxicbiocompatible water-soluble polymeric material which is capable of ionicinteraction with free amino groups in said polyamino acid to provide anouter biocompatible negatively-charged surface, said biocompatiblesemi-permeable membrane being in the form of a hydrogel having anoverall water content within the membrane structure of at least about 20wt. %, said biocompatible semi-permeable membrane being permeable to andpermitting nutrients and oxygen to flow from a body in which themicrocapsule is implanted to said living tissue of individual cellsthereof and permitting metabolic products of said living tissue to flowtherefrom to the body in which the microcapsule is implanted and beingimpermeable to said living tissue to retain the living tissue within themicrocapsule, said microcapsule being capable of resisting degradationand remaining permeable in vivo for at least two months.
 2. Themicrocapsules of claim 1 wherein said living tissue is an animal tissueselected from the group consisting of islets of Langerhans, liver andindividual cells thereof.
 3. The microcapsules of claim 1 wherein saidpolyamino acid is comprises of polylysine, the inner surface iscomprised of alginate and the outer surface is comprised of alginate,polyvinyl alcohol or polylactic acid.
 4. The microcapsules of claim 1wherein said living tissue is islets of Langerhans or a fraction thereofwhereby insulin flows from the microcapsules, and said biocompatiblesemi-permeable membrane remains effective for a period of at least threemonths on implantation of said microcapsule to control blood sugarlevels in the body in which the microcapsule is implanted.
 5. Themicrocapsule of claim 4, wherein said islets of Langerhans are suspendedin an aqueous medium.
 6. A method of encapsulating a core materialwithin a semi-permeable membrane which is a hydrogel having an overallwater content within the membrane structure of at least about 20 wt. %,which method comprises:(a) placing the material in an aqueous solutionof a water-soluble polymeric substance that can be reversibly gelled andwhich has free acid groups, (b) forming the solution into droplets, (c)gelling the droplets to produce discrete shape-retaining temporarycapsules, (d) forming biocompatible semi-permeable membranes about thetemporary capsules by contact between the temporary capsules and abiocompatible polyamino acid polymer containing free amino groups tocause ionic reaction with the acid groups in a surface layer of thecapsule to provide a positively-charged surface, and (e) contacting saidmicrocapsules formed in step (d) with a non-toxic biocompatible watersoluble polymeric material which contains free negatively-charged groupscapable of ionic reaction with the free amino groups of said polyaminoacid polymer in surface layer of the microcapsule, thereby to form anouter coating of said biocompatible polymeric material on saidmicrocapsules having a negatively-charged surface,said semi-permeablemembrane formation and said contact thereof with biocompatible polymericmaterial being such as to form microcapsules having a diameter of about50 to about 2000 μm and a semi-permeable membrane thickness of about 5to about 20 μm, and being such as to produce microcapsules capable ofresisting degradation and remaining permeable in vivo for at least twomonths.
 7. The method of claim 6 wherein said core material comprisesliving tissue which is in finely-divided suspended form in said aqueoussolution in step (a).
 8. The method of claim 7 wherein said livingtissue comprises islets of Langerhans whereby said microcapsules may beused to control blood sugar levels in diabetic animal bodies into whichthe microcapsules are implanted.
 9. The method of claim 4 wherein saidreversibly-gellable water-soluble substance is a polysaccharide gum. 10.The method of claim 6 wherein said gum is an alkali metal alginate. 11.The method of claim 6 wherein said polymer-containing free amino groupshas a molecular weight of about 11,000 to about 400,000 daltons.
 12. Themethod of claim 6 wherein said polymer containing free amino groupscomprises polylysine having a molecular weight of about 11,000 to about100,000.
 13. The method of claim 6 wherein said biocompatible polymericmaterial comprises a polysaccharide gum containing free acid groups. 14.The method of claim 6 wherein said biocompatible negatively-chargedpolymeric material is selected from the group consisting of polyvinylalcohols having free hydroxyl groups and polylactic acids containingfree acid groups.
 15. The method of claim 6 wherein saidreversibly-gellable water-soluble substance comprises sodium alginate,said polymer containing free amino groups comprises polylysine, and saidbiocompatible polymeric material comprises sodium alginate.
 16. Themethod of claim 20 wherein said alkali metal alginate is sodium alginateand the viscosity of said aqueous solution of sodium alginate is atleast sufficient to result in the formation of substantially sphericaltemporary capsules.
 17. The method of claim 16, wherein said aqueoussodium alginate solution has a viscosity of at least about 30 cps. 18.The method of claim 6 including the further step of reliquifying the gelwithin the semi-permeable membrane.